Kinetics of the conformational alterations associated with nucleophilic modification of .alpha.2-macrobglobulin

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Abstract
The effect of a nucleophilic modification of [a2macroglobulin .alpha.2M) with methylamine on the kinetics of SH exposure was investigated. The generated SH groups were detected with 4,4''-dithiodipyridine. The bimolecular rate constant for SH exposure was determined to be 11.6 .+-. 0.8 M-1 s-1 at 30.degree. C and pH 8.0. Treatment of .alpha.2M with methylamine or proteases, such as plasmin and trypsin, results in a substantial increase in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid. This probe was used to monitor the kinetics of the conformational change occurring in .alpha.2M upon treatment with methylamine. The conformational change did not occur simultaneously with the cleavage of the thiol ester bonds by the nucleophile but, rather, the conformational alterations occurred following a lag phase. The data are consistent with a mechanism requiring the random cleavage of 2 thiol ester bonds of a dimeric unit in the molecule prior to the unimolecular process representing the conformational change. According to this model, the 2 dimeric units present in .alpha.2M act as independent entities. On the basis of the best fit with the model, the bimolecular rate constant, for hydrolysis of the thiol ester bonds, was determined to be 11.9 .+-. 0.7 M-1 s-1, while the rate constant for the conformational change was (9.7 .+-. 2.0) .times. 10-3 s-1. The measured rate of conformational change is rate limited by thiol ester cleavage at lower methylamine concentrations. The conformational change, measured with this fluorescence probe, approximately parallels the loss of trypsin binding activity of .alpha.2M, measured by resistance of the bound trypsin to soybean trypsin inhibitor. A much slower loss of plasmin binding activity was observed than was found for trypsin, suggesting that the structural requirements on .alpha.2M for the interaction with plasmin are disrupted much more slowly than the structural requirements for trypsin binding upon methylamine treatment of the molecule.